PAT solutions to monitor adsorption of Tetanus Toxoid with aluminum adjuvants.

The focus of this study was to examine the small-scale adsorption process of Tetanus Toxoid (TT) as a model protein antigen to aluminum phosphate (AlPO4) and aluminum oxyhydroxide (AlOOH) adjuvants with real-time monitoring by in-line ReactIR™, ParticleTrack™ based on Focused Beam Reflectance Measurement (FBRM) and EasyViewer™ probes. The adsorption process of AlPO4 and AlOOH with TT using was monitored in the small-scale reactors. Conformational changes in TT were monitored using in-line infrared probe ReactIR, whereas particle formation associated with protein adsorption were measured by particle size, count, and imaging tools, such as ParticleTrack with FBRM and EasyViewer probes. ParticleTrack distribution results and kinetic measurements were also supported by observations made using EasyViewer. In addition to EasyMax, BioBLU reactor was also used for the adsorption experiments. ReactIR with ATR-Fiber probe was effectively able to monitor adsorption progress of TT to AlOOH and to AlPO4. ReactIR, EasyViewer, and ParticleTrack provided detailed mechanistic and kinetic information for reaction of TT with AlPO4 and AlOOH. These in-situ measurements revealed a possible multi-step process for TT to AlPO4 which may be an indication of antigen adsorption.

An intrinsic fluorescence method for the determination of protein concentration in vaccines containing aluminum salt adjuvants.

Determination of protein concentration in vaccines containing aluminum salt adjuvant typically necessitates desorption of the protein prior to analysis. Here we describe a method based on the intrinsic fluorescence of tyrosine and tryptophan that requires no desorption of proteins. Adjuvanted formulations of three model Bordetella pertussis antigens were excited at 280 nm and their emission spectra collected from 290 to 400 nm. Emission spectra of protein antigens in the presence of aluminum salt adjuvants were able to be detected, the effects of adjuvants on the spectra were analyzed, and linear regressions were calculated. The fluorescence method proved to be very sensitive with a limit of quantification between 0.4 and 4.4 µg/mL and limit of linearity between 100 and 200 µg/mL, across the formulations tested. The fluorescence method was found to be influenced by adjuvant presence, type of adjuvant, adjuvant concentration, buffer and pH conditions. The method also demonstrated ability to monitor the percent adsorption of antigens to the adjuvants. Furthermore, intrinsic fluorescence showed good correlation with micro-Kjeldahl elemental assay in quantifying protein concentration. Being a non-invasive, quick and sensitive method, intrinsic fluorescence has the potential to be utilized as a high throughput tool for vaccine development and conceivably implemented in-line, using in-line fluorimeters, to monitor antigen concentration during formulation processing.

A high ratio of IC31(®) adjuvant to antigen is necessary for H4 TB vaccine immunomodulation.

A tuberculosis (TB) vaccine consisting of a recombinant fusion protein (H4) and a novel TLR9 adjuvant (IC31) is in clinical development. To better understand the H4-IC31 ratio, we measured the binding capacity of IC31 for H4 protein and immunized mice with formulations that contained limiting to excess ratios of IC31 to H4. An immunomodulated H4-specific IFNγ response was only observed when IC31 was present in excess of H4. Since TLR expression is species-specific and the vaccine is intended to boost BCG-primed immunity, we questioned whether data in mice would translate to humans. To address this question, we used the fresh human Whole Blood (hWB) recovered from BCG-vaccinated subjects to screen H4-IC31 formulations. We found IC31 modulation in hWB to be quite distinct from the TLR4-Adjuvant. Unlike TLR4-Adjuvant, IC31 formulations did not induce the pro-inflammatory cytokine TNFα, but modulated a robust H4-specific IFNγ response after 12 d of culture. We then re-stimulated the fresh hWB of 5 BCG-primed subjects with formulations that had excess or limiting IC31 binding for H4 protein and again found that an immunomodulated H4-specific IFNγ response needed an excess of IC31. Finally, we monitored the zeta (ζ) potential of H4-IC31 formulations and found that the overall charge of H4-IC31 particles changes from negative to positive once IC31 is in greater than 9-fold excess. Using two diverse yet mutually supportive approaches, we confirm the need for an excess of IC31 adjuvant in H4 TB vaccine formulations and suggest surface potential may be an important factor.